Cooperative binding of estrogen receptor to DNA depends on spacing of binding sites, flanking sequence, and ligand.

It has been suggested that cooperative binding of estrogen receptor (ER) may, in part, be responsible for the synergistic activation of transcription of estrogen-responsive genes that contain multiple estrogen-response elements (EREs). Experiments described here show that estradiol-liganded ER (E2-ER) binds cooperatively to stereoaligned EREs that are surrounded by naturally occurring flanking sequences, such as an AT-rich region. In contrast, EREs lacking these sequences do not bind E2-ER cooperatively, regardless of ERE spacing or stereoalignment. Moreover, binding is of lower affinity and capacity in the absence of these critical flanking sequences. By varying the sequence of nucleotides adjacent to the ERE, features important for the flanking sequence effect were characterized. Interestingly, when ER was liganded with 4-hydroxytamoxifen (4-OHT), the active metabolite of the widely used therapeutic antiestrogen tamoxifen, the antiestrogen-liganded ER complex (4-OHT-ER) did not bind cooperatively to multiple EREs, regardless of spacing or flanking sequence. We postulate that ERE flanking sequences bestow upon E2-ER enhanced ERE binding capacity and cooperativity, but do not affect 4-OHT-ER-ERE binding.